Therapeutic Potential of a Synthetic Lethal Interaction Between the MYC Proto-Oncogene and Inhibition of Aurora-B Kinase

Therapeutic potential of a synthetic lethal interaction between the MYC proto-oncogene and inhibition of aurora-B kinase

Dun Yanga, Hong Liua, Andrei Gogab, Suwon Kimc, Mariia Yunevaa, and J. Michael Bishopa,1

aDepartment of Microbiology and Immunology, G. W. Hooper Research Foundation, and bDivision of Hematology/Oncology, Department of Medicine, University of California, San Francisco, CA 94143; and cDepartment of Basic Medical Sciences, College of Medicine, University of Arizona, Phoenix, AZ 85004

Contributed by J. Michael Bishop, June 17, 2010 (sent for review May 4, 2010) The Myc protein and proteins that participate in mitosis represent Results attractive targets for cancer therapy. However, their potential is Synthetic Lethal Interaction Between MYC and VX-680. To elucidate presently compromised by the threat of side effects and by a lack of the cellular response to VX-680, we examined the effect of the drug pharmacological inhibitors of Myc. Here we report that a circum- on human retinal pigment epithelial cells that expressed either the scribed exposure to the aurora kinase inhibitor, VX-680, selectively neomycin resistance gene (RPE-NEO) or NEO plus an ectopic kills cells that overexpress Myc. This synthetic lethal interaction allele of human MYC (RPE-MYC) (Fig. 1A and Fig. S1). Over the is attributable to inhibition of aurora-B kinase, with consequent course of 7–9 d, continuous application of VX-680 (300 nM) killed disabling of the chromosomal passenger protein complex (CPPC) the entire population of RPE-MYC cells. Moreover, the killing and ensuing DNA replication in the absence of cell division; exe- proceeded to completion even if VX-680 was withdrawn after 3 d cuted by sequential apoptosis and autophagy; not reliant on the (Fig. 1A and Fig. S1B). tumor suppressor protein p53; and effective against mouse models Withdrawal of the drug was accompanied by prompt reac- MYC for B-cell and T-cell lymphomas initiated by transgenes of .Our tivation of aurora-B in the cells (Fig. 1B). In contrast, RPE-NEO fi ndings cast light on how inhibitors of aurora-B kinase may kill cells were not killed by VX-680. Instead, the cells failed to pro- tumor cells, implicate Myc in the induction of a lethal form of auto- liferate in the presence of VX-680, but doubled in number within phagy, indicate that expression of Myc be a useful biomarker for 6 d after withdrawal of the drug (Fig. 1A and Fig. S1B). A similar MEDICAL SCIENCES sensitivity of tumor cells to inhibition of the CPPC, dramatize the arrest of proliferation without death in response to VX-680 was virtue of bimodal killing by a single therapeutic agent, and suggest observed with other rodent and human cells that do not over- a therapeutic strategy for killing tumor cells that overexpress Myc express Myc (Table S1). while sparing normal cells. Initial Lethal Response to VX-680 Is Apoptotic Death. Exposure to apoptosis | aurora kinase | autophagy synthetic lethality | targeted VX-680 for 3 d killed ∼30% of RPE-MYC cells (Fig. 1A and Fig. therapy | chromosomal passenger protein complex S1A). By the end of the 3-d treatment, virtually all of the surviving cells had become multinucleated and polyploid, and had entered he MYC proto-oncogene encodes a bHLHzip-type transcrip- a new phase of killing (see below). Thus, we divided the killing by Ttion factor (Myc) that is a key regulator of cell growth, pro- VX-680 into two phases, defined as “early” (death within 3 d) and liferation, and survival (1). Overexpression of Myc transforms “delayed” (all subsequent deaths). VX-680 caused both early and various cells in vitro, causes tumors in mice, and ranks among the late death of RPE-MYC cells and other cells that overexpressed more common tumorigenic anomalies found in a wide variety of ectopic Myc, including mouse embryo fibroblasts (MEF-MYC), human malignancies (1, 2). Although these features make Myc rat embryo fibroblasts (Rat1A-MYC), and human kidney epi- a potential target for cancer therapy, practicable inhibition of the thelial cells (HA1E-MYC) (Table S1). protein with pharmaceuticals has yet to be achieved. An alter- Early cell death was associated with activation of the canonical native approach is to exploit synthetic lethal interactions between form of apoptosis in RPE-MYC, Rat1A-MYC, and HA1E-MYC overexpression of Myc and the therapeutic induction of other cells (Fig. 1C, Fig. S2A,andTable S1). The fraction of cells affected cellular aberrations that do not kill normal cells (3–5). by apoptosis could account for at least two-thirds of the early cell VX-680 is a potent and reversible small-molecule inhibitor of deaths (Fig. S2A). Apoptosis first became apparent by day 2 of the aurora family of mitotic kinases (6). Among the targets for exposure to VX-680, reached a peak at day 3, and faded with the VX-680 is aurora-B kinase/AURK B, the catalytic subunit of the inception of the delayed phase of death (Fig. S2B). We do not know chromosomal passenger protein complex (CPPC), which also why apoptosis was limited to a minority of the cell population, but contains the proteins survivin/BIRC5, INCENP, and Borealin this limit was seen in mouse, rat, and human cells (Table S1). (7). The complex is involved in multiple facets of cell division, We demonstrated that apoptosis was responsible for the con- including the spindle checkpoint, chromosome segregation, and current cell death by two means. First, we examined the proa- cytokinesis (7). Here we report a synthetic lethal interaction poptotic protein Bim, which is known to mediate apoptosis in the between VX-680 and Myc overexpression. This effect can be presence of excess Myc (8). Bim was vigorously expressed in RPE- achieved by any means that inhibits or depletes the aurora-B MYC cells but was undetectable in RPE-NEO cells (Fig. S2C). Par- kinase or otherwise disables the CPPC. The synthetic lethality is tial depletion of Bim with RNAi (Fig. S2C) provided substantial mediated mainly by a combination of apoptosis and lethal autophagy. It kills cells that overexpress Myc in vitro while fi sparing normal cells, and it has therapeutic ef cacy when tested Author contributions: D.Y. and J.M.B. designed research; D.Y. and H.L. performed re- in mice bearing tumors elicited by a transgene of MYC.We search; A.G., S.K., and M.Y. contributed new reagents/analytic tools; D.Y. and J.M.B. suggest that overexpression of Myc might represent a biomarker analyzed data; and D.Y. and J.M.B. wrote the paper. for identifying tumors that are likely to respond to treatment The authors declare no conflict of interest. with inhibitors of aurora-B kinase at a favorable therapeutic 1To whom correspondence should be addressed. E-mail: [email protected]. index. Moreover, the bimodal nature of cell killing may provide This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a deterrent to the emergence of drug resistance. 1073/pnas.1008366107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1008366107 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Fig. 1. VX-680 elicits apoptosis and autophagy in cells that overexpress Myc. (A) Cell killing by VX-680. RPE-NEO cells (□ and ■) and RPE-MYC cells (○ and ●) were treated with 300 nM VX-680 for 3 d and then either transferred into drug-free medium (○ and □) or maintained in fresh medium containing VX- 680 (● and ■). Cells were harvested at daily intervals and then assayed for viability by the trypan blue exclusion assay. The data are expressed as mean ± SD for three independent experiments. The arrow indicates the point at Fig. 2. VX-680 elicits polyploidy in cells that overexpress Myc. (A) The effect which VX-680 treatment was terminated. (B) Reversible inhibition of aurora- of VX-680 on DNA synthesis. RPE-NEO cells (□) and RPE-MYC cells (■) were B kinase by VX-680. RPE-MYC cells were treated for 3 d with 300 nM VX-680 treated with 300 nM VX-680 for 3 d and then transferred into drug-free and then transferred into drug-free medium. Cells were fixed either before (i medium. BrdU incorporation assays were performed at the indicated times and iii) or 3 h after (ii and iv) the transfer and stained for DNA in blue (iii and after initiation of VX-680 treatment. Each column, here and in B, represents iv) and phosphorylation of histone H3 at Ser-10 in green (i and ii), a surro- the average of three independent experiments, and each experiment was gate assay for the activity of aurora-B kinase. (C) Activation of caspase-9. done in triplicate. Error bars represent 1 SD. #Cells below the limit of de- RPE-MYC cells were treated for 3 d with either 0.03% DMSO or 300 nM tection. (B) The effect of VX-680 on the mitotic index. RPE-NEO cells (□)and VX-680, fixed, and stained for DNA (blue) and active caspase-9 (red). (D) RPE-MYC cells (■) were treated with 300 nM VX-680 for 0 or 3 d, fixed, and Ultrastructural analysis of cell morphology. RPE-MYC cells were treated with stained for DNA. The mitotic index was expressed as the percentage of cells in 300 nM VX-680 for 3 d and then transferred into drug-free medium. Rep- mitosis. (C) VX-680 elicits abnormalities in centrosome number, centromere resentative electron micrographs of RPE-MYC cells either before (a)or7 number, and the mitotic spindle. RPE-MYC was treated with 300 nM VX-680 d after (b–d) administration of VX-680 are shown. M, mitochondria; N, nu- for 3 d and then stained for centrosomes with anti-pericentrin antibodies in cleus. White arrows denote vacuoles containing electron-dense material in green (a, an untreated RPE-MYC cell; b, a treated RPE-MYC cell), centromeres b, multimembrane vesicles in c, and localized cytoplasmic degeneration in d. with the CREST human autoserum in red (c), microtubules with anti–α-tubulin Black arrowheads in c and d point to double-membrane vesicles sequester- antibodies in red (d), and DNA with DAPI in blue (a–d). A representative cell is ing a portion of cytoplasm. shown in each panel. (Scale bar: 5 μm.) (D) The effect of VX-680 on centrosome number. The percentage of mitotic RPE-MYC cells with the indicated number of centrosomes (numbered squares) was determined after various durations of treatment with 300 nM VX-680. A representative experiment is protection against the early death elicited by VX-680 (Fig. S2D). shown. (E) The effect of VX-680 on cell morphology. RPE-MYC cells were Second, we found that the pancaspase inhibitor z-VAD-fmk effec- treated with either 0.03% DMSO (a and c) or 300 nM VX-680 (b and d)for3d ted a two-thirds reduction in early cell death (Fig. S2E). and then photographed. Three-fold magnifications of a field of RPE-NEO cells To further characterize the selectivity of early cell killing by and a single multinucleated RPE-MYC cell are shown in c and d, respectively. (Scale bar: 20 μm.) (F) Polyploidy elicited by VX-680 in cells that overexpress VX-680, we surveyed a variety of genes that have been impli- Myc. RPE-NEO cells and RPE-MYC cells were treated with either DMSO or cated in tumorigenesis. To date, we have observed synthetic le- 300 nM VX-680 for the indicated days, and DNA content was analyzed by thality with MYCN, the E6 oncogene of human papillomavirus FACS after staining DNA with 10 μg/mL of propidium iodide. (G) Over- 16, the E1A gene of adenovirus, and NOTCH1-IC, which enc- expression of Myc prevents induction of the p53-dependent G1 checkpoint odes a constitutively active version of the Notch protein (Fig. pathway. RPE-NEO cells and RPE-MYC cells were treated for the indicated time periods with 300 nM VX-680, and expression of the indicated proteins S3A). MYCN is closely related to MYC and has been implicated was assessed by Western blot analysis. Equal loading was confirmed by in a synthetic lethal interaction with inhibition of the CDK2 ki- staining of total proteins on the transfer membrane with Ponceau S. nase (9). In accordance with previous reports (10–12), E6, E1A, and NOTCH1-IC all induced vigorous expression of Myc in the RPE-NEO test cells (Fig. S3B), which provides a credible ex- Myc Overexpression Permits Continuation of DNA Replication During planation for their synthetic lethal interaction with VX-680. Arrested Cytokinesis. VX-680 arrests cells late in cytokinesis (6), Deficiencies in the tumor suppressor proteins p53 and Rb were which normally would lead to arrest of DNA replication (13–15). not synthetic lethal with VX-680 (Fig. S3C). However, during a 3-d treatment with VX-680, RPE-MYC cells

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1008366107 Yang et al. Downloaded by guest on September 29, 2021 continued DNA replication (Fig. 2A)andmitosis(Fig.2B), steadily increased their numbers of centrosomes and chromosomes (Fig. 2 C and D), and became enlarged, multinucleated, and polyploid (Fig. 2 E and F). By day 10, no cells remained, however (Fig. 1A and Fig. S1B). In contrast, the same treatment caused RPE-NEO cells to arrest DNA synthesis and mitosis (Fig. 2 A and B), but not before ∼70% of the cells had become tetraploid (Fig. 2F), presumably due to inhibition of the spindle checkpoint by aurora-B kinase (14). DNA synthesis was renewed when VX-680 was removed from RPE- NEO cells after 3 d (Fig. 2A), in accordance with the revival of cellular proliferation (Fig. 1A and Fig. S1B). By day 10, ∼75% of the cells were actively synthesizing DNA (Fig. 2A). The arrest of DNA synthesis elicited by disablement of the CPPC in normal cells is mediated by the p53-p21 pathway (13– 15). Accordingly, the amounts of p53 and p21 increased in RPE- NEO cells treated with VX-680 (Fig. 2G). In contrast, the constitutive expression of p21 in RPE-MYC cells was greatly di- minished compared with that in RPE-NEO cells (Fig. 2G). Moreover, although treatment with VX-680 elicited a modest induction of p53 in RPE-MYC cells, it produced no notable change in the expression of p21 (Fig. 2G). It appears that activation of the p53- p21 pathwaywas blocked when Myc was overexpressed, asdescribed previously (16), accounting for the failure of VX-680 to arrest Fig. 3. The autophagy genes ATG5andATG7 contribute to delayed cell death DNA synthesis. elicited by VX-680. (A) Depletion of Atg5 and Atg7 by their cognate RNAi. RPE- The cellular response to cytotoxic agents also is typically medi- MYC cells were infected with either pLKO.1-puro control lentiviral particles or ated by the p53-p21 pathway (17), and a deficiency in this pathway is lentiviral particles encoding shRNA against either ATG5 or ATG7, selected with a common means through which tumor cells become resistant to 0.5 μg/mL of puromycin for 2 d, and subjected to Western blot analysis for Atg7 cytotoxic drugs (18). In contrast, such a deficiency did not create and Atg5, which are found mainly as Atg5–Atg12 conjugates. Actin was used as resistance to the synthetic lethal interaction between VX-680 and a loading control. (B) Suppression of autophagy by depletion of either Atg5 or MEDICAL SCIENCES Myc in either model cell lines (Table S1)orhumantumorcelllines Atg7. RPE-MYC cells were stably transfected with Cherry-LC3, infected with the indicated RNAi viruses, treated with 300 nM VX-680 for 3 d, and then transferred (Fig. S4). We conclude that the synthetic lethal interaction between fi VX-680 and Myc does not require the p53-p21 pathway—avalu- into drug-free medium. Quanti cation of autophagy with Cherry-LC3 was per- fi formed 2 d after withdrawal of VX-680. Cells that had more than 40 Cherry-LC3 able property, given the frequency at which that pathway is de cient puncta per cell were scored as positive. Each column, here and in ensuing panels, in human tumors (18). represents the average of three independent experiments, and each experiment was done in triplicate. Error bars represent SD. (C and D) Depletion of autophagy Myc Overexpression Elicits Autophagy in Response to VX-680. Apo- proteins suppresses delayed cell death, but not early death. RPE-MYC cells were ptosis in RPE-MYC cells was not readily detectable during infected with the indicated RNAi lentiviruses, treated with 300 nM VX-680 for delayed death. Instead, the cells displayed features of autophagy 3 d, and then transferred into drug-free medium. Cells were harvested at 0, 3, (Fig. 1D and Fig. S5 A–F). This phenotype first became apparent and 6 d after initiation of VX-680 administration and assayed for viability by the at 3 d after initiation of treatment with VX-680 (Fig. S5 A, B, D, trypan blue exclusion assay. (C) Delayed death was calculated using the fol- − and F), reached a maximum by 5 d, and eventually affected at least lowing formula: percentage of dead cells = [(live cell number at day 3 live cell number at day 6)/(live cell number at day 3)] × 100. (D) Early death was expressed 80% of the cells (Fig. S5 B and D), even though the drug had been as the percentage of dead cells at day 3. (E and F) Absence of atg5 protects cells − − withdrawn after 3 d. RPE-NEO cells did not respond to VX-680 in against delayed death, but not against early death. atg5+/+ and atg5 / MEF cells this manner (Fig. S5 A, B, and D–F). were infected with either a control virus (pMig) or a Myc-expressing virus We suspected that the interaction between VX-680 and Myc (pMigMyc). The infected cells were then treated with DMSO or 300 nM VX-680 might induce the expression of genes responsible for autophagy. for 3 d and transferred into drug-free medium. Cells were harvested at 0, 3, and We focused on ATG6andATG9, because these genes are involved 6 d after initiation of treatment with VX-680 and assayed for viability by the in initiating the genesis of autophagosomes (19). We found that trypan blue exclusion assay. Early death (E)anddelayeddeath(F)werecalcu- # treatment with VX-680 augmented the expression of both ATG6 lated as described for C and D. Cells below the limit of detection. In the ex- μ and ATG9 in RPE-MYC cells, but not in RPE-NEO cells (Fig. periment represented by the hatched column, 100 M z-VAD-fmk was included in the medium during the delayed cell death assay. S5A). Induction of the ATG genes became evident within 2 d after administration of VX-680, when autophagy was still at constitutive Second, we used mouse embryo fibroblasts established from atg5+/+ levels, and reached a maximum within 5 d, congruent with the peak − − of autophagy (Fig. S5 A, B, D,andF). We conclude that Myc and and atg5 / mice (20). Ectopic overexpression of Myc rendered both VX-680 induce ATG6andATG9 in a collaborative manner, and the WT and null cells susceptible to early death elicited by VX-680 that this induction is probably integral to the augmentation of (Fig. 3E). In contrast, the absence of atg5provided>2-fold greater autophagy by the synthetic lethal interaction. protection against delayed death (Fig. 3F). We conclude that a substantial portion of the delayed cell death Autophagy Is a Major Effector of Delayed Death. The congruence of was executed by autophagy. However, neither depletion of Atg autophagy with delayed death raised the possibility that autophagy proteins by RNAi nor a genetic deficiency in atg5 provided complete was the agency of delayed death. We addressed this possibility in protection against delayed death. The residual death was not likely twoways.First,weusedRNAitodepletecellsofeitherAtg5orAtg7 the product of apoptosis, because it could not be blocked by z-VAD- protein (Fig. 3A), two essential components of the canonical auto- fmk (Fig. 3F). A possible explanation is the action of a noncanonical phagy pathway (19). These depletions greatly suppressed autophagy pathway to autophagy that is independent of Atg5 and Atg7 (21). (Fig. 3B) and provided substantial, but incomplete, protection against the delayed death of RPE-MYC cells in response to VX-680 Synthetic Lethal Interaction Between VX-680 and Myc Overexpression (Fig. 3C). In contrast, depletion of the Atg proteins provided no Is Due to Disablement of the CPPC. The pharmacological effects of protection against the early, apoptotic form of death (Fig. 3D). VX-680 are not limited to aurora kinases (22). We used two

Yang et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 with VX-680. We tested this possibility by either growing RPE- MYC cells to conﬂuence or treating these cells with aphidicolin before exposing them to VX-680. Both procedures arrested DNA synthesis (Fig. S6 A and B) and substantially protected the cells from both early and delayed killing by VX-680 (Fig. S6 C–F). The elevated expression of Myc in RPE-MYC cells was not affected by either conﬂuence or aphidicolin (Fig. S6G). We conclude that sustained DNA synthesis is essential for the synthetic lethal interaction between Myc and VX-680, perhaps by virtue of the ensuing polyploidy or DNA damage (Discussion).

Therapeutic Efficacy of VX-680 Against Myc-Driven Models of Lym- phoma. Our studies in vitro indicated that VX-680 might be useful in the treatment of tumors that overexpress Myc. We explored this possibility with mouse models that develop either B-cell (24) or T-cell (25) lymphomas in response to Myc overexpression. The B-cell tumors are driven by a constitutively active transgene of MYC (24), whereas the transgene responsible for the T-cell tumors can be repressed by doxycycline (25). We used pulse therapy to exploit the fact that the synthetic lethal interaction continues to kill cells after withdrawal of VX-680, whereas at Fig. 4. Synthetic lethal interaction between disablement of the CPPC and least some fraction of normal cells may recover from the effects overexpression of Myc. (A) Cell killing by AZD1152. RPE-NEO cells (□ and ■)and of the drug. RPE-MYC cells (○ and ●) were treated with 50 nM AZD1152 for 3 d and then ○ □ Treatment with VX-680 was initiated at 7 d after cells from either transferred into drug-free medium ( and ) or maintained in fresh primary tumors were transplanted to naive isogenic mice, by which medium containing VX-680 (● and ■). Cells were harvested at the indicated intervals and then assayed for viability by the trypan blue exclusion assay. The point enlargement of the liver and spleen was inevitably apparent. data are expressed as mean ± SD for three independent experiments. The arrow Intermittent pulses of treatment were continued until more than indicates the time at which VX-680 treatment was terminated. (B) Depletion of half of the animals were deceased (Fig. 5 B and C). The result was the chromosomal passenger proteins survivin, aurora-B, and INCENP by RNAi. an ∼3-fold increase in survival. Treatment of primary T-cell tumors Extracts from RPE-MYC cells treated with RNAi against luciferase, INCENP, sur- led to a prompt and major reduction in phosphorylation of histone viving, or aurora-B were analyzed by immunoblotting for expression of survivin, H3 at Ser-10 at a dose that proved effective against both B-cell and aurora-B, and INCENP at 4 d after transfection with RNAi. (C) Early apoptosis T-cell tumors (75 mg/kg body weight) (Fig. 5A), demonstrating that elicited by depletion of chromosomal passenger proteins in cells that overexpress Myc. Apoptosis was assessed by DAPI staining of DNA at 4 d after the drug inhibited aurora-B kinase in vivo, consistent with the in- transfection of RPE-NEO cells (□) and RPE-MYC cells (■) with RNAi against the hibition being responsible for the tumor response. indicated genes. Cells with fragmented nuclei were scored as positive for apo- A single 4-d treatment of animals bearing the transplanted ptosis. Each column, here and in D, represents the average of three independent T-cell lymphoma shrank the spleen, liver and kidney (Fig. 5D and experiments, and each experiment was done in triplicate. Error bars represent 1 Table S2), and greatly reduced the number of infiltrating tumor SD. (D) Autophagy elicited by depletion of chromosomal passenger proteins in cells in nonlymphoid organs such as liver and kidney (Fig. 5E). A cells that overexpress Myc. Autophagy was assessed by immunostaining for LC3 single 5-d treatment of animals bearing a primary T-cell lymphoma □ ■ at 7 d after transfection of RPE-NEO cells ( ) and RPE-MYC cells ( )withRNAi increased the amount of apoptosis in tumors 4- to 5-fold (Fig. 5F against the indicated genes. Cells thathad morethan40 LC3 puncta per cell were scored as positive. (E and F) Delayed cell death elicited by depletion of the and Fig. S7A) and the number of polyploid cells by 8-fold (Fig. 5F chromosomal passenger proteins. RPE-MYC cells (E) and RPE-NEO cells (F)were and Fig. S7B), and elicited autophagy in tumor tissue (Fig. 5G). replated at 5% and 20% confluence, respectively, at 4 d after transfection with Mice subjected to the intermittent treatment described in Fig. RNAi against indicated genes. The depletions by RNAi were comparable to those 5B gained weight (Fig. S7C) and demonstrated little if any change illustrated in B. Cells were photographed after being fixed and stained with in peripheral blood cells (Fig S7D). In contrast, daily treatment for crystal violet at 4 d (E)or7d(F) after replating. 16 d caused a modicum of weight loss and a 7-fold reduction in neutrophils, in accordance with a previous report (6). In addition, approaches to examine whether the killing of cells by VX-680 a 5-d treatment with VX-680 had no appreciable effect on weight, was due to inhibition of aurora-B kinase and consequent dis- histology, apoptosis, or ploidy in the spleens of normal mice (Fig. ablement of the CPPC. First, we exploited another inhibitor of 5F and Fig. S7 A and B). We conclude that a synthetic lethal in- aurora kinases, AZD1152 (23), which affects aurora-B and -C, teraction between VX-680 and Myc overexpression can be elicited but not aurora-A. AZD1152 replicated the early and delayed in vivo with consequent therapeutic efficacy, and that the thera- killing of RPE-MYC cells described above for VX-680 (Fig. 4A). peutic regimen that we used was well tolerated. Second, we demonstrated that depletion of aurora-B, survivin, and INCENP with RNAi (Fig. 4B) elicited apoptosis (Fig. 4C), Discussion autophagy (Fig. 4D), and delayed death that eliminated virtually We have found that a circumscribed exposure to the aurora ki- all of the remaining cells (Fig. 4E). In contrast, RPE-NEO cells nase inhibitor VX-680 selectively kills cells that overexpress Myc. recovered from each of the depletions (Fig. 4F) as the effect of The synthetic lethal interaction is demonstrable in experimental RNAi decayed over time. We conclude that both the early and cell lines that overexpress ectopic MYC, human tumor cell lines delayed cell death elicited by VX-680 can be attributed to dis- that overexpress intrinsic MYC or MYCN, and murine tumors ablement of the CPPC by inhibition of aurora-B kinase, and that initiated by transgenes of MYC. The killing results from prompt any other form of such disablement likely would also display apoptosis and delayed autophagy, is not compromised by with- a synthetic lethal interaction with overexpression of Myc. drawal of the drug after 3 d of treatment, and has demonstrable therapeutic efficacy in animal models. We were able to demon- DNA Synthesis Is Required for the Synthetic Lethal Interaction Between strate that aurora-B kinase is the crucial target for VX-680 in Myc and VX-680. It seemed possible to us that illicit DNA synthesis both the early and the delayed killing, and that the lethal re- was the trigger for the synthetic lethality in RPE-MYC cells treated sponse can be attributed to disablement of the CPPC.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1008366107 Yang et al. Downloaded by guest on September 29, 2021 Molecular Path to Synthetic Lethality. It is possible to postulate a sequence of events that gives rise to the bimodal synthetic lethal interaction between Myc and disablement of the CPPC. The sequence begins when cytokinesis is arrested by failure of the CPPC. In the presence of excess Myc, the G1/S checkpoint is compromised and illicit DNA synthesis ensues, leading to polyploidy in the absence of cytokinesis. Inhibition of aurora-B causes the spindle checkpoint to fail, resulting in multinucleation. Myc overexpression may contribute to this mayhem in any of several ways, including direct promotion of DNA replication (26) and circumvention of both the G1/S and G2/M checkpoints (16, 27, 28). The end result is an unleashing of the proapoptotic activity of Myc (29) and induction of ATG genes by a demonstrable (albeit yet unexplained) collaboration between Myc and VX-680. Thus, the cells that survive the wave of apoptotic killing are destined to die later, due mainly to lethal autophagy. The illicit DNA synthesis that occurs during the synthetic lethal interaction might well give rise to DNA damage (30, 31). The cyto- static and apoptotic responses to DNA damage are typically mediated by p53 (17) and overexpression of Myc channels that respond to apoptosis (16). But the synthetic lethal interaction does not require p53, so DNA damage may not be the trigger for the interaction. Instead, the polyploidy resulting from illicit DNA synthesis might be responsible (32). There has been uncertainty about the role of p53 in the cellular response to VX-680 and other inhibitors of aurora-B kinase (33, 34). The uncertainty may arise in part from unappreciated variables. For Fig. 5. VX-680 extends the survival of mice with lymphomas elicited by Myc. example, a previous study found that VX-680 induces apoptosis (A) Inhibition in vivo of aurora-B kinase in tumor cells. Tet-o-MYC/Eμ-SR-tTA preferentially in cells with defective p53 function (34), whereas in transgenic mice maintained on a doxycycline-free diet were treated with 0 mg/kg our experience, deficiencies in p53 were not synthetic lethal with MEDICAL SCIENCES (lane 1), 45 mg/kg (lane 2), 65 mg/kg (lane 3), and 75 mg/kg (lane 4) of VX-680 via VX-680 (Table S1 and Fig. S3C). At least two factors could account i.p. injection when tumors became evident. Higher doses were precluded by lim- for this discrepancy. First, the previous study used HPV-E6 to in- ited solubility of VX-680. Single-cell suspensions were prepared from spleen at 6 h activate p53 in cells with the wild-type allele of TP53, and we found after injection and analyzed for histone H3 and its phosphorylation at Ser-10 by Western blot analysis. (B) Survival of mice bearing T-cell lymphomas. Isogenic naïve that the E6 gene itself has a synthetic lethal interaction with VX-680, mice were transplanted with T-cell lymphoma cells (1 million cells per recipient) presumably mediated by induction of Myc. Second, the p53-null from a single Tet-o-MYC/Eμ-SR-tTA transgenic mouse. One week later, the recip- cells that responded to VX-680 in that study are known to over- ients were treated with either vehicle (n = 20) or 75 mg/kg of VX-680 (n = 10) by express intrinsic Myc. daily i.p. injection at the times indicated by arrows (see SI Materials and Methods for details). Survival is displayed as a Kaplan-Meier plot. Survival curves for vehicle Synthetic Lethal Approaches to Cancer Therapy. Previous reports treatment and VX-680 treatment were compared using the log-rank test; P = have described synthetic lethal interactions between Myc and 0.0001. (C) Survival of mice bearing B-cell lymphomas. Isogenic naïve mice un- various external agents; examples include activation of the DR5 derwent transplantation with B-cell lymphoma cells (50,000 per recipient) from μ death receptor pathway with the TRAIL ligand (3), inhibition of asingleE -MYC mouse and treated as described in B. Survival curves for vehicle cyclin-dependent kinase CDK1 (4), and the antimalarial drug treatment and VX-680 treatment were compared using the log-rank test; P = 0.0001. (D and E) Tumor suppression by VX-680. Isogenic naïve mice underwent chloroquine (5). In addition, inhibition of CDK2 is synthetically transplantation with T-cell lymphomas (1 million cells per animal) and treated with lethal with overexpression of MycN (9). The synthetic lethal effect either vehicle or 75 mg/kg of VX-680 for 4 d by daily i.p. injection that was initiated described here has several advantageous features, however. 1 wk after transplantation. Six days after the final injection, the indicated internal First, cytotoxic therapies typically elicit apoptosis. The bi- organs from animals were harvested for photography (D) and H&E staining of modal nature of the synthetic lethality that we have described fixed sections (E). (F) VX-680 elicits apoptosis and polyploidy in MYC-driven lym- may permit the treatment of cancer cells that are defective in phomas in vivo. Tet-o-MYC/Eμ-tTA transgenic mice maintained on a doxycycline- apoptosis, a common circumstance that can confer resistance to free diet were treated with five daily i.p. injections of 75 mg/kg of VX-680 or ve- fi therapy (18). For example, we have encountered a number of hicle when tumors became evident (Dox OFF). One day after the nal injection, human tumor cell lines that overexpress either MYC or MYCN spleens were removed and fixed with 4% paraformaldehyde. Spleens for controls were obtained from Tet-o-MYC/Eu-tTA mice that had been kept tumor-free by the and are defective in apoptosis, yet were killed by the delayed administration of doxycycline in the diet (Dox ON). Sections of spleens were an- response to VX-680 (Table S3). Second, the strategy described alyzed by H&E staining, TUNEL assay, and propidium iodide staining. Black and here targets proteins expressed only in dividing cells, and it might white arrows indicate polyploid cells. (G) Induction of autophagy by VX-680 in have minimal toxicity for nondividing cells. Finally, the cytotoxic vivo. Six Tet-o-MYC/Eμ-tTA transgenic mice were treated with vehicle (lanes 1 and effect of VX-680 on proliferating cells might be minimalized by 2) or five daily i.p. injections of 75 mg/kg of VX-680 (lanes 3–6) when tumors be- administering the agent in circumscribed pulses, which can kill came evident. Single-cell suspensions were prepared from spleen at 24 h after the tumor cells that overexpress Myc. It is notable that normal mice last injection and assessed by Western blot analysis with antibodies against LC3 appeared to tolerate pulsed treatment with VX-680 well. and actin. The numbers indicate the ratio between LC3-II and LC3-I. The Myc-selective killing of cells by chloroquine involved both apoptosis and autophagy (5), but in a manner different from what Functions upstream of Myc in cell signaling pathways might we have described here. Treatment with chloroquine induced large- display similar synthetic lethality. NOTCH1-IC, HPV-E6, and ade- scale autophagy but impaired its terminal steps, resulting in induc- novirus E1A provide examples, because they can induce the ex- tion of p53-dependent apoptosis. Cell death was not impaired by a pression of Myc and display synthetic lethality with VX-680 in RPE- genetic deficiency of atg7, and an unexplained form of killing oc- NEO cells (Fig. S3A). It should be worthwhile to pursue other such curred when apoptosis was blocked. Chloroquine appeared to examples, in the hope of amplifying the number of therapeutic tar- be effective as a preventive agent in a mouse model for B-cell gets that might display synthetic lethality with inhibitors of the CPPC. lymphoma, but demonstrated no efficacy as a therapeutic.

Yang et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 We conclude that overexpression of Myc and genes that activate Materials and Methods MYC are potential biomarkers for sensitivity of tumor cells to VX- See SI Materials and Methods for details on all materials and procedures, 680 or any other agent that compromises the CPPC. There may well including (i) cell lines, transfections, and retroviral transductions; (ii) assays be genes that are capable of a similar synthetic interaction without for cellular proliferation, DNA synthesis, cell cycle, apoptosis, and autophagy; the intervention of Myc, but we have yet to uncover an example of (iii) chemical and immunologic reagents; (iv) imaging procedures; (v) mouse such genes. The synthetic lethal interaction between Myc and VX- strains, tumorigenesis, and treatment protocols; (vi) analysis of cellular RNA 680 may create a favorable therapeutic index, and may be valuable and proteins; (vii) the use of RNAi; and (viii) statistical analyses. as a biomarker in the design of both future clinical trials and ther- ACKNOWLEDGMENTS. We thank Gerard Evan, David Morgan, and Scott apeutic regimens. Preclinical studies of combination therapy ex- Kogan for assistance with the manuscript; Jay Debnath (University of ploiting two or more of the several synthetic lethal interactions with California, San Francisco) for the Cherry-LC3 construct; Robert Weinberg Myc that have now been defined also merit consideration. (Massachusetts Institute of Technology) for HA1E cells; Noboru Mizushima (Tokyo Medical and Dental University) for the atg5−/− MEF cells; and Tyler − − Jacks (Massachusetts Institute of Technology) for the Trp53 / MEF cells. We Note Added in Proof. Subsequent to review of the manuscript and also thank Ivy Hsieh and Juan Engel for excellent support with transmission final submission to the editor, an online publication reported that electron microscopy and Jane Gordon for confocal laser microscopy. This re- inhibition of aurora-B kinase selectively killed cells that overexpress search was supported by the Susan G. Komen Breast Cancer Research Fund (D. Myc and displayed efficacy in a preclinical model for B-cell lym- Y.), a Howard Hughes Medical Institute Physician-Scientist Fellowship and National Institutes of Health Grant K08 CA104032 (to A.G.), National Cancer phoma (Blood, doi 10.1182/blood-2009-11-251074). The bimodal Institute Grant K01 CA115681 (to S.K.), and funds from the G.W. Hooper nature of the killing reported here was not described. Research Foundation (to J.M.B.).

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